Dynamic Self-Assembly and Stimuli-Responsive Disassembly of Bioactive-Loaded Cubosomes in Biomimetic Media Traced by Real-Time Small-Angle X-ray Scattering and Cryogenic Transmission Electron Microscopy

Designing advanced functional materials capable of passing through complex biological environments requires a deep understanding of their dynamic structural behavior <i>in situ</i>. We investigate pH-responsive core-shell cubosomes for oral drug delivery applications. These nanoparticles comprise a lipid-based core of cubic <i>Im3m</i> liquid crystalline structure and are coated with a chitosan-<i>N</i>-arginine/alginate polyelectrolyte shell (PS). The cubosomes encapsulate varying concentrations (0-30% w/w) of <i>Aloe vera</i>-derived acemannan, an immunomodulatory macromolecular drug. Utilizing synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we performed an advanced spatiotemporal analysis, which focused on their nanoscale structural evolution under simulated gastric (pH 2.5) and intestinal (pH 7.4) conditions. The interactions with key individual gastrointestinal components, including mucins, pepsin, bile salts, and pancreatin, were systematically examined. Our results demonstrate that acemannan incorporation and environmental pH significantly modulate cubosome structure and heterogeneity (phase coexistence) during disassembly. The pH-responsive polyelectrolyte shell imparts notable structural stability against pepsin and mucins at pH 2.5, ensuring functional gastric protection. However, under intestinal conditions (pH 7.4), bile salt-mediated solubilization caused complete disassembly. Pancreatic lipase-induced digestion triggered a remarkable time-dependent phase transition from a cubic (<i>Im3m</i>) to an inverted hexagonal (H_II) topology in PS-cubosomes containing 30% acemannan. A simulated duodenum mixture induced lamellar phases at pH 2.5 for acemannan-loaded systems but led to complete disassembly at pH 7.4, primarily driven by bile salts. Deconvoluting these structural responses over time provides crucial insights into their mechanistic nature. It clarifies pH-dependent stability and component-specific disassembly pathways. The achieved understanding is crucial for designing advanced stimuli-responsive lipid/biopolymer nanomaterials that facilitate efficient oral delivery.